Although hyperphosphatemia is deemed a risk factor of the progression of chronic kidney disease (CKD), it remains unclear whether the normal range of serum phosphorus likewise deteriorates CKD. A propensity score analysis was applied to examine the causal effect of the normal range of serum phosphorus on the incidence of end-stage renal disease (ESRD).A retrospective CKD cohort of 803 participants in a single institution was analyzed. Propensity score was estimated using 22 baseline covariates by multivariate binary logistic regression for the different thresholds of time-averaged phosphorus (TA-P) in the normal range of serum phosphorus incremented by 0.1 mg/dL from 3.3 to 4.5 mg/dL.The incidence rate of ESRD was 33.9 per 1,000 person-years over median follow-up of 4.3 years. Total patients showed the mean baseline phosphorus of 3.37 mg/dL and were divided to quartile. The higher quartile was associated with the parameters consistent with the advancement of CKD. A stratified Cox regression showed the highest hazard ratio (HR) at TA-P 3.4 mg/dL  adjusted for baseline covariates such as sex, age, diabetic nephropathy, estimated GFR, serum albumin, Na-Cl, phosphorus, LDL-C and proteinuria. Adjusted HRs remained high up to TA-P 4.2 mg/dL . After propensity score matching conducted at the thresholds of TA-P 3.4, 3.6, 3.8 and 4.0 mg/dL, the higher levels of TA-P showed the higher HRs by Kaplan-Meier analysis (p < 0.05 by stratified log-rank test). The numbers needed to treat were calculated as 3.9 to 5.3 over 5 years.The propensity score analysis shows that even the normal range of serum phosphorus clearly accelerates CKD progression to ESRD. Our results encourage clinicians to target serum phosphorus to inhibit CKD progression in the manner of ‘the lower the better.’ Early recognition and intervention against risk factors responsible for the progression of chronic kidney disease (CKD) are expected to improve renal outcomes of patients at risk . Major r2.Most recently we have found that the higher normal range of serum phosphorus, either at baseline or in the follow-up, may be a strong risk factor of CKD progression towards ESRD , 5. A raTo explore this hypothesis, we conducted a standard survival analysis using serum phosphorus in the follow-up and a propensity score-based survival analysis, the latter of which is increasingly being used to estimate causal effects in the observational studies because one can replicate the prospective randomized controlled trial by minimizing baseline confounding . We utilWe used a retrospective CKD cohort already reported n = 803) , 5, 8, a03 , 5, 83), albumin-corrected calcium (cCa), inorganic phosphorus (P), low-density lipoprotein cholesterol (LDL-C) and C-reactive protein (CRP). Urine parameters included spot urine proteinuria (expressed as gram per gram creatinine excretion) and spot urine hematuria by dipstick . Due to the distribution, C-reactive protein, proteinuria and hematuria were log-transformed for analyses.The demographic characteristics included sex, age, body mass index (BMI), original kidney disease (diabetic nephropathy or not) and systolic blood pressure (SBP). Blood parameters included hemoglobin (Hb), white blood cell (WBC), platelet (Plt), albumin (Alb), uric acid (UA), sodium (Na), potassium (K), chloride (Cl), Na-Cl  by trapezoidal rule  depending on the distribution. The relationship between baseline phosphorus and TA-P were tested by Pearson’s correlation analysis. C-statistics for the accuracy of the propensity score models were obtained by the area under the receiver operating characteristic (AUROC) curve for the threshold . Hosmer-ropriate . Standarropriate , 20. Forropriate . StatistDuring the follow-up period of median 4.3 [interquartile range 2.6–5.7] years, 110 out of 803 patients progressed to ESRD. The incidence rate was 33.9 per 1,000 person-years. The mean baseline phosphorus was 3.37 ± 0.52 mg/dL. The distribution of baseline phosphorus was almost normal as depicted in p < 0.001). The regression line between TA-P versus baseline phosphorus was less than a unity in the slope and converged at 3.9 mg/dL of phosphorus due to the increase in serum phosphorus in the follow-up in the lower phosphorus groups.Next, the distribution of the TA-P was examined and is also depicted in per se could not provide target range of phosphorus to inhibit CKD progression. Also, the impacts of the time-varying parameter on CKD progression in the early stage and the late stage of the clinical course vary with time thus the time-averaged value is not appropriate for the risk factor analysis.A time-to-event survival analysis was performed with TA-P for predicting the incidence of ESRD. TA-P was divided by a threshold from 3.3 to 4.5 mg/dL by a 0.1 mg/dL increment. A dichotomous value of TA-P thus obtained and other baseline covariates were tested for proportional hazards assumption, which turned out not violated. Multicollinearity was not observed, either. Crude HRs and its 95% confidence interval (CI) are plotted in p < 0.05 in some situations . These results were consistent with those of the stratified multivariate Cox proportional hazards models  and the shorter observation period (median 2.3 vs. 4.3 years) as compared with our cohort. Accordingly, an overall incidence rate of ESRD was 8.1 per 1,000 person-years much less than 33.9 in the present study. Therefore, the extended observation period of their study may be warranted.Worth mentioning is that no independent association of serum phosphorus with risk for CKD progression to ESRD was reported recently . Mean phper se may exacerbate kidney dysfunction [The most striking finding in the present study is that such a low concentration of serum phosphorus as 3.4 mg/dl, well within the normal range, can be a risk factor of the progression of CKD to ESRD. The mechanism by which higher phosphorus can aggravate kidney injury may be partially extrapolated to this clinical scenario as suggested previously –37 but tfunction , 38. Secfunction . In any We have to mention about limitations of the present study. The first problem is the potential presence of unmeasured confounders which cannot be avoided in any observation study. Next, there is a possibility of misspecification of the propensity score model which cannot be asserted by any means, either. We believe the latter problem could be solved by accommodating a stratified multivariate Cox proportional hazards model as herein demonstrated. Lastly, this study was not a therapeutic trial and not designed to assess the impact of serum phosphorus targeting to lower normal range on the progression of CKD. Despite these limitations, a propensity score analysis clearly captures its unbounded potential to examine many test conditions such as risk factors and target thresholds. Needless to say, randomized controlled trials remain the gold standard to build evidence while a propensity score analysis may serve complementary approaches in the clinical research on the causal effect .The propensity score analysis unveiled that even normal levels of serum phosphorus in the follow-up can predict the risk of ESRD. Target range of serum phosphorus in the follow-up may be less than 4.3 mg/dL to inhibit the progression of CKD to ESRD with “the lower the better” principle. The results may facilitate vigorous treatment for lowering serum phosphorus level in clinical practice. The mechanism whereby normal range of serum phosphorus render kidney injury in the setting of CKD should be elucidated in animal and human studies.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.


Escherichia coli adhering to surfaces makes type-1 fimbriae stretch out like springs to catch on to mannosidic receptors. This mechanism is initiated by a disruption of the quaternary interactions between the lectin and the pilin of the two-domain FimH adhesin and transduces allosterically to the mannose-binding pocket of FimH to increase its affinity. Mannose-specific adhesion of 14 E. coli pathovars was measured under flow, using surface plasmon resonance detection on functionalized graphene-coated gold interfaces. Increasing the shear had important differential consequences on bacterial adhesion. Adherent-invasive E. coli, isolated from the feces and biopsies of Crohn’s disease patients, consistently changed their adhesion behavior less under shear and displayed lower SPR signals, compared to E. coli opportunistically infecting the urinary tract, intestines or loci of knee and hip prostheses. We exemplified this further with the extreme behaviors of the reference strains UTI89 and LF82. Whereas their FimA major pilins have identical sequences, FimH of LF82 E. coli is marked by the Thr158Pro mutation. Positioned in the inter-domain region known to carry hot spots of mutations in E. coli pathotypes, residue 158 is indicated to play a structural role in the allosteric regulation of type-1 fimbriae-mediated bacterial adhesion.Shear force exerted on uropathogenic  Escherichia coli (E. coli) species express type-1 pili or fimbriae, which are organelles integral to host receptor recognition and attachment [N-terminal mannose-binding lectin domain connected by a flexible linker to a pilin anchoring it to FimG and FimF in the tip fibrillum. Natural variations in amino acid composition were found to regulate the pathoadaptivity of E. coli [E. coli type-1 fimbriae  from theceptors . This coeceptors  or of theceptors ,8.E. coli type-1 fimbriae to the extensibility of the stubby and flexible tip fibrillum [E. coli to mannose-coated surfaces in laminar flow chambers is enhanced by shear stress [E. coli pathotypes [E. coli experience shear stress. Therefore, we decided to examine 14 E. coli clinical isolates, from a variety of niches in the human host, including the urinary tract, the intestine and loci of infections in knee and hip prostheses, for their capability to adhere under increasing shear stress.Here, we demonstrate the importance of amino acid variations in FimH on the capacity of the whole FimH TDA to extend its structure, adopt the high-affinity conformation and initiate fimbrial stretching and further uncoiling . Early eibrillum . Thomas r stress . These ar stress . The Fimr stress . The domr stress . Evidencr stress , structur stress ,14,15 anthotypes , no struE. coli [Shear is the dominant effect of fluid flow and can be controlled by changing the flow rate through microfluidic channels. The drag force exerted per surface area is called shear or shear stress (τ). Fimbriated bacteria can relieve some of the shear stress by spreading their fimbrial organelles and pulling themselves closer to the plasmonic interface . When exE. coli ,21.E. coli on Au interfaces modified with carbohydrates can be followed with very high sensitivity using surface plasmon resonance (SPR) detection [E. coli UTI89 can be conveniently studied by following the SPR signals on the graphene-coated plasmonic interfaces [It was recently demonstrated that the growth of etection . We haveterfaces ,24. Grapterfaces , and graterfaces , for theterfaces  and for terfaces  interactterfaces ,30,31.d-mannopyranoside (HM) are taken advantage of here [E. coli pathovars for the comparison of their adhesion under quasi-identical experimental conditions. Remarkably, a consistently lower threshold to withstand shear stress was observed for adherent-invasive E. coli (AIEC) isolates from Crohn’s disease patients compared to other E. coli pathotypes. The most affected AIEC strains contain mutations in the inter-domain region of the FimH TDA  [d-mannopyranoside (3), nitrophenyl α-d-glucopyranoside (4) and nitrophenyl β-d-glucopyranoside (5) were synthesized as reported [2SO4 solution [Au-graphene biosensors were prepared as described in the ide (HM) , nitrophreported . The Au-solution  (Table SE. coli strains (600nm) of around 10 as the starting concentration for hemagglutination. A spectrophotometrically-obtained optical density measured at 600 nm of 1.0 conforms to on average 0.66 × 109 colony-forming units (cfu)·mL−1. The same bacterial preparations were used for hemagglutination and SPR experiments. Each E. coli strain was cultured at least twice under these same conditions.Each of the  strains  has beenE. coli strains. The E. coli strains, with a starting concentration as determined by spectrophotometry, were two-fold diluted in PBS, and 50 µL of each dilution were deposited in round-bottomed 96-well plates. The red blood cells were washed twice in the ice-cold PBS, diluted to 5% of the original solution, and 50 µL were added to the wells. The plates were left on ice for one night before read-out of the hemagglutination titer.Guinea pig erythrocytes  were agglutinated by the different −1. The bacterial strain solutions were diluted to 1 × 108 cfu/mL in the running buffer PBS and injected at different flow rates, 5, 10, 20, 30, 50 and 100 µL·min−1, corresponding to 5, 10, 30 and 100 mPa, respectively, into the two flow channels. At the 30-min time point after injection of bacteria, the bacterial load on the surface was stable for all flow rates  equipped with a laser light of a 650-nm wavelength and two channels that are each served by 60-µL loops. The channels are 12-mm long and have an inner diameter of 0.5 mm, which allow laminar flow (Re = 3) at speeds from 4 to 100 µL·minow rates , and thefimH gene was amplified in PCR reactions using colonies as the template. The primers used to amplify fimH were uti8+4913338 and uti8-4914820 [® Gel and PCR Clean up, Macherey-Nagel, Düren, Germany), then submitted for Sanger sequencing by GATC Biotech, Konstanz, Germany. Sequencing primers were fimhZLfp ATGAAACGAGTTATTACCCTGT, fimhZLrp TTGATAAACAAAAGTCACGCCAAT, Uti8+4913927 CCGGTGGCGCTTTATTTGAC and Uti8+4913338. Sequence comparisons were done using the BLASTN 2.3.1+ program at NCBI [The -4914820  .The overlays and comparisons of crystal structures were performed using the graphics program Coot  and PymoE. coli with host epithelial receptors is mediated by the FimH two-domain adhesin (TDAs), positioned at the tip of multimeric hair-like appendages, called type-1 fimbriae [E. coli (at a concentration of 108 cfu·mL−1) and carbohydrate-coated Au-graphene interfaces (d-mannoside (HM) was identified in this work as a prototype ligand for the modification of Au-graphene, because the presence of the amino end group allows for a tight control of carbohydrate loading by X-ray photon spectroscopy (XPS) analysis  and the corresponding β-anomer (5) were used as negative controls. XPS analyses indicated that Au-graphene is less loaded with mannose (1) and methyl α-d-mannoside (2) than with aminoheptyl α-d-mannoside (HM) and nitrophenyl α-d-glucosides (4), in agreement with the degree of carbohydrate loading quantified by using an optimized Molisch test [E. coli UTI89  modified interfaces displayed largely decreased SPR responses when compared to the aminoheptyl and nitrophenyl analogues. This might have partially resulted from the lower carbohydrate loading for the latter , a range used by others [E. coli UTI89 at six different flow rates. An increase in flow rate will in the first instance stimulate encounters of the microbes with the substrate due to an increase in microbial transport [−1  mutant FimH that proves totally dysfunctional in terms of mannose binding [E. coli strains under flow. Having established this concept, one may now start to distinguish molecular properties of FimH variants that allosterically regulate adhesion behavior under shear stress from those permitting the binding of mannose.To validate more closely the carbohydrate-specific component in the  binding ,36,48. BE. coli clinical isolates from human patients was investigated to gain a better understanding of pathogen interactions at the initial stage of infection. The fimH genes of the newly isolated E. coli strains from osteoarticular infections were sequenced and compared to the amino acid sequence of the laboratory E. coli strain K12 MG1655  (The adhesion behavior of 2 MG1655 . Togetheiopsies) .E. coli strains with Au-graphene-HM as a function of flow rate. They confirm that a minimal shear (>5 µL·min−1) is needed to observe increased adhesion, as evidently, the flow rate is known to increase the adhesion rates in microfluidic devices [−1) than for the other disease-associated E. coli strains, where higher flow rates (up to 30 µL·min−1) result in further increased bacterial interaction with the HM-modified SPR interface  indicates the absence of mannose-sensitive type-1 fimbriae on the bacterial surface. This is the case for two E. coli isolates from osteoarticular infections and one from the feces of a Crohn’s disease patient  that does not vary much with the flow rate, although a light shear force-dependent binding pattern is visible  is less enhanced under shear force than for the other tested E. coli strains, and the maximal enhancement is already attained at a lower flow rate. The AIEC strain LF82 demonstrates this at the extreme, by displaying a quasi-shear force-independent adhesion with an already optimal affinity under static conditions. The high-affinity state of FimH has been associated with the elongated conformation of the fimbrial tip TDA  that are 100-fold higher than for α-d-mannose (Kd = 2 μM) [E. coli in the urinary tract [E. coli LF82 (The FimH adhesin has affinities for mannotriose and mannopentaose (K = 2 μM) ,63. A hiry tract . Strangery tract , howeveroli LF82 .E. coli clinical isolates. The interfaces could easily be modified with aminoheptyl α-d-mannopyranoside, a ligand with an affinity (Kd = 20 nM) just like mannotriose, to study specific binding of E. coli under flow. The low influence of shear stress on the adhesion capacity of AIEC strains may indicate that in the gut, enteric E. coli have the opportunity to bind glycan receptors with a structure based on the mannotriosidic core of natural oligomannosides [N-linked glycans, as is known in the bladder [Au-graphene interfaces modified with high-affinity ligands for FimH have here shown potential as plasmonic surfaces to obtain information on laminar flow-enhanced interactions of nnosides , rather  bladder . FurtherE. coli, such as AIEC, to enhance affinity through a shear-dependent mechanism. It remains hitherto unknown whether any of the other mutations found in the inter-domain region of the FimH adhesins of Crohn’s-associated AIEC strains [E. coli pathovars. The reduced capacity of AIEC strains to relieve shear stress and prevent bacterial detachment may be well due to the different physico-mechanical (shear) and chemical (glycan receptor) conditions imposed on them during the colonization of epithelial linings. AIEC colonize the thick, mucosal layers of the intestinal epithelium, whereas uropathogenic E. coli bind the highly glycosylated urothelial cells that undergo large shear during voiding and filling of the bladder. A lack of dynamism in response to shear may also render AIEC more susceptible to soluble inhibitors, such as anti-adhesives that counteract the FimH adhesin [The presence of mannotriosidic receptors for FimH may have eliminated the need for enteric  strains  (Figure  adhesin . In conc


High genetic heterogeneity in the hepatitis C virus (HCV) is the major challenge of the development of an effective vaccine. Existing studies for developing HCV vaccines have mainly focused on T-cell immune response. However, identification of linear B-cell epitopes that can stimulate B-cell response is one of the major tasks of peptide-based vaccine development. Owing to the variability in B-cell epitope length, the prediction of B-cell epitopes is much more complex than that of T-cell epitopes. Furthermore, the motifs of linear B-cell epitopes in different pathogens are quite different (e. g. HCV and hepatitis B virus). To cope with this challenge, this work aims to propose an HCV-customized sequence-based prediction method to identify B-cell epitopes of HCV.This work establishes an experimentally verified dataset comprising the B-cell response of HCV dataset consisting of 774 linear B-cell epitopes and 774 non B-cell epitopes from the Immune Epitope Database. An interpretable rule mining system of B-cell epitopes (IRMS-BE) is proposed to select informative physicochemical properties (PCPs) and then extracts several if-then rule-based knowledge for identifying B-cell epitopes. A web server Bcell-HCV was implemented using an SVM with the 34 informative PCPs, which achieved a training accuracy of 79.7% and test accuracy of 70.7% better than the SVM-based methods for identifying B-cell epitopes of HCV and the two general-purpose methods. This work performs advanced analysis of the 34 informative properties, and the results indicate that the most effective property is the alpha-helix structure of epitopes, which influences the connection between host cells and the E2 proteins of HCV. Furthermore, 12 interpretable rules are acquired from top-five PCPs and achieve a sensitivity of 75.6% and specificity of 71.3%. Finally, a conserved promising vaccine candidate, PDREMVLYQE, is identified for inclusion in a vaccine against HCV.http://e045.life.nctu.edu.tw/BcellHCV.This work proposes an interpretable rule mining system IRMS-BE for extracting interpretable rules using informative physicochemical properties and a web server Bcell-HCV for predicting linear B-cell epitopes of HCV. IRMS-BE may also apply to predict B-cell epitopes for other viruses, which benefits the improvement of vaccines development of these viruses without significant modification. Bcell-HCV is useful for identifying B-cell epitopes of HCV antigen to help vaccine development, which is available at  Infection with the hepatitis C virus (HCV) often results in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma . HCV prein silico analysis of HCV epitopes using algorithms to predict the immunogenicity of their variants from other less studied genotypes  before being input into the SVM.Physicochemical properties (PCPs), also referred to as propensity, are the most intuitive feature associated with biochemical reactions and are widely used in the field of bioinformatics. This work represents each peptide sequence for an  AAindex  databasep/aaindex. In thisC and γ of the used SVM simultaneously based on an inheritable bi-objective genetic algorithm  using the composition of PCPsStep 1) Each sample is represented as an i ffrom which to select PCP features and two 4-bit genes for encoding kernel parameter (γ) and cost parameter (C). The corresponding feature i p(the i-th PCP feature) is excluded from the SVM classifier if i f= 0, and is included if i f= 1. Let m be the sum of if. The γ > 0 determines how the samples are transformed into a high-dimensional search space. The cost parameter C>0 of the SVM classifier adjusts the penalty of total error. These two parameters C and γ must be tuned to get the best prediction performance. In this work, γ ∈ {2-15, 2-13, ..., 216}and C∈ {2-15, 2-13, ..., 216}.Step 2) The IBCGA-chromosome consists of binary genes m selected PCP features, γ and C by decoding the IBCGA-chromosome. In this work, a popular kernel function that is radial basis function exp (− γ ||i xjx||2) is adopted. The i xand j xare training samples and γ is a kernel parameter. The parameter settings of IBCGA are given in Additional File Step 3) The fitness function is the prediction accuracy of 10-fold CV using the LIBSVM classifier  with m sr Sfrom r=start rto rend are obtained using IBCGA. Let m Sbe the most accurate solution with m selected PCP features among all solutions from C to C search space. In this work, start r= 10 and rend = 40 are used.Step 4) All solutions for R independent runs to obtain the best R number of discrete runs to get the best R solutions, In this work, R = 30 is used.Step 5) IBCGA use mechanisms of randomization and are therefore characterized as non-deterministic based on the fact that results of individual runs are not the same always. Therefore, Steps 3) and Step 4) are performed for Decision tree algorithms are valuable to obtain the rule-based knowledge since the tree can generate if-then rules. In this work, the method C5.0 is employed to construct decision tree classifier and acquire interpretable rule set for analyzing hepatitis C virus. The decision tree is constructed using ranked properties, which selected by information gain, and can be used to select properties, according to the ranks of properties. Nevertheless, the selected properties have no interaction between each property and the influence of properties should be considered individually. For acquiring the general and interpretable rules, the pruning process is applied to avoid the over-fitting problem and the threshold value of confidence is set to 25%. The final tree can transfer to if-then rules that one rule is corresponded to one leaf node. The covered samples of a rule are the samples in one leaf node.C, γ) of SVM classifier in this study. The 34 informative physicochemical properties were selected by the PCP mining training module.To provide prediction service to the scientific community, we have developed a user-friendly web server Bcell-HCV based on 34 informative physicochemical properties and optimized parameters  and Ming-Ju Tsai (MJT) designed the system, participated in manuscript preparation, and carried out the detail work. MJT, Kai-Ti Hsu, Jyun-Rong Wang (JRW) and Yi-Hsiung Chen (YHC) designed the system and implemented programs. Also, Shinn-Ying Ho (SYH) and WLH supervised the whole project and participated in manuscript preparation. All authors have read and approved the final manuscript.Table S1. Definition of the 34 properties ranked by the accuracy differences.Click here for fileTable S2. Rule-based knowledge of Bcell-HCV prediction.Click here for fileTable S3. Statistics of the top-k epitopes for the threshold > 0.95.Click here for fileTable S4. The top-50 B-cell epitopes of HCV for analyzing the conserved motif and constructing a phylogenetic tree are listed. The order of peptide IDs are sorted using the prediction score.Click here for fileFigure S1. Figure S1 (a) HCV source organism. (b) A total of 8009 non-redundant linear B-cell epitopes and non B-cell epitopes are obtained.Click here for fileFigure S2. Figure S2 Collect relevant antigenic sequences with (b) B-cell assay data, based on the (b) positive samples and (c) negative samples.Click here for fileTable S5. The control parameters of IBCGA used.Click here for file


Multidrug-resistant tuberculosis (MDR-TB) drugs which is resistant to the major first-line anti-TB drugs, Isoniazid and Rifampicin, has become a major global challenge in tuberculosis (TB) control programme. However, its burden at community level is not well known. Thus, the aim of study was to assess the prevalence of primary and secondary resistance to any first line anti-TB drugs and MDR TB in Hitossa District of Oromia Regional State, Central Ethiopia.Population based cross- sectional study was conducted on individuals aged ≥15 years. Those with symptoms suggestive of TB were interviewed and two sputum specimens were collected from each and examined using Lowenstein-Jensen (LJ) culture medium. Further, the isolates were confirmed by the Ziehl-Neelsen microscopic examination method. Drug susceptibility test (DST) was also conducted on LJ medium using a simplified indirect proportion method. The resistance strains were then determined by percentage of colonies that grew on the critical concentration of Isoniazid, Streptomycin, Rifampicin and Ethambutol.The overall resistance of all forms of TB to any first-line anti-TB drug was 21.7 %. Of the total new and previously treated culture positive TB cases, 15.3 and 48.8 % respectively were found to be a resistant to any of the first-line anti-TB drugs. Further, of all forms of TB, the overall resistance of MDR-TB was 4.7 %. However, of the total new TB cases, 2.4 % had primary while 14.3 % had secondary MDR-TB. Resistance to any of the first-line anti-TB drugs (adjusted odd ratio (AOR), 8.1; 95 % CI: 2.26–29.30) and MDR-TB (AOR), 7.1; 95 % CI: 2.6–43.8) was found to be linked with previous history of anti-TB treatment.The study has identified a high rate of primary and secondary resistance to any of the first-line anti-TB drugs and MDR-TB in the study area. The resistance may have resulted from sub-optimal performance of directly observed treatment short-course (DOTS) programme in the detecting infectious TB cases and cure rates in the study area. Anti-TB drug resistance is linked with previous TB treatment. There is a need to strengthen DOTS and DOTS-Plus programmes and expand MDR-TB diagnostic facilities in order to timely diagnose MDR-TB cases and provide appropriate treatment to prevent the spread of MDR-TB in Ethiopia.The online version of this article (doi:10.1186/s12889-016-3210-y) contains supplementary material, which is available to authorized users. Tuberculosis (TB) remains a high-priority communicable disease that causes illness among millions of people and is the second leading cause of death from an infectious disease worldwide . The disMultidrug-resistant tuberculosis (MDR-TB) is defined as Mycobacterium tuberculosis strain resistant to at least the first-line anti-TB drugs of Isoniazid and Rifampicin. . MDR-TB Multidrug-resistant TB has been known to be a major challenge in TB control programme. It has been spreading rapidly across the globe, and in recent years an estimated 3.5 % of new cases and 20.5 % of previously treated TB cases have MDR-TB. In 2013, there were an estimated 480,000 MDR-TB cases, and about 210,000 deaths were caused by MDR-TB worldwide .th out of the 27 countries with the highest estimated number of multidrug resistant tuberculosis (MDR-TB) cases. Additionally, WHO estimated the prevalence of MDR-TB among new TB cases in Ethiopia at 1.6 % , and among the previously treated ones at 12 % (95 % CI: 5.6 to 21 %) [According to the 2014 World Health Organization (WHO) report, Ethiopia ranked 15to 21 %)  and secoto 21 %) , 12, 13.Nevertheless, to our knowledge, reports from Ethiopia on the prevalence of MDR-TB including primary and secondary resistance were based on reports among health service seekers from health facilities and might therefore be subject to selection bias –16.Because of the absence of population-based studies and adequate laboratory testing facilities in the country, it is difficult to have a reliable estimation on the burden of primary and secondary resistance to any first line anti-TB drugs and MDR TB. However, this population-based study conducted in Hitossa District of Arsi zone Oromia Regional State, Central Ethiopia was the first attempt in the country aimed at measuring the prevalence of primary and secondary resistance to any first line anti-TB drugs and MDR-TB.kebele  [kebeles have been further divided into three sub-kebeles known as gote. Each gote has about 1/3 population of the kebele [The study was conducted in Hitossa District of Arsi Zone, Oromia Regional State in Central Ethiopia. The population density, socio–economic state, and demographic condition of Hitossa are typical representative of the entire features of Arsi Zone. The Directly Observed Treatment, Short-Course (DOTS) and health service coverage, the proportion of urban–rural population and HIV prevalence, and the TB case notification and treatment outcomes of Hitossa District are also similar to those of the whole zone of Arsi. The district has an estimated population of 178,229 people living in 23 rural and one urban kebeles were included in the study. One gote from each kebele was randomly selected, and individuals 15 years and above living in the households of the selected gote were enrolled in the study. A week before the baseline survey, pre-survey registration was carried out in the selected gotes of the 24 kebeles. Accordingly, 61,678 individuals living in 9,454 households were identified. Eligibility includes permanent residents of 15 years of age and above who could provide written consent for willingness to participate in the study and temporary visitors who arrived at least 15 days before the commencement of the study. Using the above criteria, 33,073 adults were identified and included in the study.Population based cross sectional study was conducted between 1 July 2013 and 30 June 2014 to estimate the prevalence of primary and secondary multi-drug resistant to any first line anti-TB drugs and MDR-TB in Hitossa District of Arsi Zone. For practical and economic reasons Hitossa District was selected purposefully and all its kebele leaders. A total of 24 teams with one nurse and one health extension worker (HEW) were involved in the data collection. Furthermore, five laboratory technicians also took part in sputum sample collection. The whole process of data collection was supervised by 10 health officers. All laboratory technicians and data collectors were selected from public health institutions in the study district, and they were trained on TB screening techniques and on how to collect and transport sputum specimen. The same team of data collectors was assigned to each kebele for both pre-survey registration and data collection. The survey was carried out three times at interval of 6 months: the first at baseline, the second at the end of the sixth month, and the third at the end of the 12th month between 1 July 2013 and 30 June 2014.The aims of the study and the procedures of data collection were discussed with zonal, district and House to house visits were carried out to identify individuals with persistent cough of more than two weeks, fever, and loss of appetite, weight loss, blood-stained sputum and chest pain or difficulty of breathing which were considered as symptoms suggestive of pulmonary TB. Individuals with such symptoms were interviewed on their socio–economic and demographic information and current and previous history of TB treatment. Subsequently, participants with any symptom suggestive of the illness were asked to submit two adequate recently-discharged mucoid or muco-purulent sputum specimens (spot-morning). The laboratory technicians collected the sputum in sterile falcon tubes and immediately placed them in a cold box at 4 °C, and transported them on the same day to Adama Regional Research Center Laboratory.Mycobacterium tuberculosis complex. The cultures were considered to be negative if no colonies were identified after 8 weeks of incubation. Moreover, the isolates from the LJ were confirmed by microscopic examination for the presence of Acid-Fast Bacillus (AFB) using Ziehl-Neelsen method. Moreover, susceptibility test of all isolates to spara-nitrobenzoic acid was carried out to identify Mycobacterium tuberculosis complex from environmental mycobacteria. However, all isolates were found to be Mycobacterium tuberculosis complex [On the following day of the sputum receipt, the morning specimen was digested and decontaminated by the standard Acetyl L-cysteine (NALC)-NaOH method , followe complex .Drug susceptibility tests(DST) were carried out using the simplified indirect proportion method on LJ medium . The proTo ensure data quality, the principal investigator and supervisors closely monitored the data collection process in which a standardized and pre-tested questionnaire was used. The collected data were coded and double entered into Epi-info version 7 statistical software by trained data clerks. Then they were checked against the original information for missing variables. Errors were corrected by referring to the original questionnaire.The percentage of drug resistance to any of the drugs or its combination with other drugs was determined. The primary resistance was calculated by dividing the number of resistant isolates among new TB cases by the total number of new TB cases found to be culture positive for a particular drug or combination of drugs multiplied by 100. Similarly, percentage of secondary resistance was calculated by dividing the number of resistant isolates in previously treated TB cases by the total number of re-treated TB cases found to be culture positive for that particular drug or combination of drugs multiplied by 100. We excluded 20 contaminated cultures from the analysis as their results were not known.Analysis was made using IBM SPSS version 20 statistical software (SPSS Inc. Chicago. 2007). Descriptive analysis was made and frequencies and odds ratios (OR) with the 95 % confidence intervals (CI) were calculated. Logistic regression analysis was used to evaluate the association between drug resistance as outcome and others related independent variables. A significance level of <0.05 was considered statistically significant. We also compare the result of current study with those of other studies carried out in Ethiopia between 1984 and 2015 to understand the trend of drug resistance over time  were males and 28,048 (84.8 %) were rural residents. The age of the respondents ranged from 15 to 94 years, with a mean age of 42.3 (±18.4 SD) years. Out of the eligible individuals, 2,758 (8.3 %) reported to have symptoms suggestive of PTB. Among these, 1,717 (62.3 %) were females and 1041 (37.7 %) were males. Of the 2,758 suspected cases, 2, 218 (80.4 %) were new, and 540 (19.6 %) had previously been treated with first-line anti-TB drugs  were new, whereas 21 (19.8 %) were previously treated patients with first-line anti-TB drugs  were found to be culture positive for M. tuberculosis isolates, primary resistant strains to any first-line anti-TB drugs were observed in 13 (15.3 %) patients. Of these, 8 (9.4 %) were resistant to each INH and STM, 4 (4.7 %) to RIF and 3 (3.5 %) to EMB. Primary MDR-TB was detected in 2 (2.4 %) strains  patients. Of these, 6 (28.6 %), were resistant to INH, 5 (23.8 %) to STM, and 4 (19 %) to RIF. Secondary MDR-TB was detected in 3 (14.3 %) isolates. Of these, one (4.8 %) was found to be resistant to all first-line drugs , 8.1; 95 % CI: 2.3–29.3) more likely to develop resistance to any first-line anti-TB drugs compared to those with no history of previous TB treatment. Similarly, urban residents were four times  more likely to have resistance to any first-line anti-TB drugs compared to their rural counterparts. Individuals who had pervious history of TB treatment were nearly seven times more likely ) to have MDR-TB compared to those who had no history of pervious exposure to anti-TB drugs (Table A total of 18 studies that had been published between 1994 and 2015 on the primary and secondary anti-TB drug resistance were reviewed. Results showed that primary resistance to any drug raged from 10.7 % in 2009 to 30.1 % in 2012. Moreover, the rate of primary MDR-TB increased from 0.6 in 1994 to 3.7 % in 2009 while that of secondary resistance to any anti-TB drugs varied from 11.1 % in 1996/7 to 85.7 % in 2005/6. According to reports from the same studies, the rate of secondary MDR-TB ranged from 15.7 % in 1996/7 and 60.8 % in 2005/6 (Table Fifteen percent of the newly diagnosed, and fifty two percent of the previously treated TB cases were resistant to one or more of the first-line anti-TB drugs. MDR TB among new cases was 2.4 %, and 14.3 % among previously treated patients. Drug resistance was associated with previous history of TB treatment and urban residence. Resistances to any one or more of first-line anti-TB drugs and MDR-TB in the study population were high.In this study, the 15 % resistance to any of the first-line anti-TB drugs among new TB cases is comparable to reports from Addis Ababa and Northern Ethiopia , 20. HowThe difference in resistance rate to one or more first-line anti-TB drugs among new and previously treated TB patients across different study settings could be attributed to the variation in TB control programme performance, study population, sample size and study methods that were used across different geographical settings. For instance, the study subjects from Addis Ababa who had high rate of resistance stain , 13 wereMoreover, previous studies from Ethiopia and elsewhere in Africa were restricted to health service seekers at health facilities. Thus, study results from such segment of population may not indicate the real burden of the disease at community level as compared to the present population-based study. Likewise, the difference in burden of the resistance cases could be due to the time of the study; in the past the prevalence of resistant strains which might be a potential source of infection was not as high as they are today. For instance, in 2001, resistance to any first-line anti-TB drug among previously treated TB patients in Arsi Zone was 31.6 % . HoweverM. tuberculosis strains [In the present study, previously treated TB cases were eight times more likely to have resistance to any of the first-line anti-TB drugs compared to new ones. The high level of anti-TB drug resistance among previously treated TB cases might have resulted from poor adherence and follow up or inadequate drug supply. This might result in selection of spontaneous mutation of  strains . Hence, Moreover, resistance to any of the first-line anti-TB drugs was higher among urban residents when compared to their rural counterparts. This is in agreement with findings from a previous study in eastern Ethiopia which reported resistance of 81.7 % among urban dwellers 18.3 % among rural residents , 40. MorThe 2.4 % prevalence of MDR-TB among the newly diagnosed TB cases in this study is very high compared to the 0 % reported in 2001 from the same area . NeverthThe 14 % prevalence of MDR-TB among previously treated TB cases obtained in this study is lower than 18 % reported by the national surveillance , the 20.In this study, previously treated TB cases were more likely to have MDR-TB than the new ones. This is consistent with a meta-analysis in sub-Saharan Africa and a systematic review in Europe where a pooled risk of MDR-TB was higher among the previously treated TB cases as compared to the new ones , 44. TheThe DOTS strategy aims to detect 70 % of infectious TB cases and achieve 85 % cure rate in order to interrupt transmission, reduce mortality and avert the emergence of drug resistance , 46. HowAlthough DOTS- plus strategy is believed to be the best strategy to prevent emergence of MDR TB by accessing presumptive MDR-TB cases to diagnostic and appropriate treatment, MDR-TB patients are challenged by much more toxic and complicated treatment of longer duration which results in poor treatment outcome and emergence of XDR-TB. Therefore, effective implementation of DOTS and DOTS-plus strategies which are believed to be a corner stone in the prevention of the emergence and spread of MDR-TB and XDR-TB should be strengthen in the country. Thus, expanding MDR-TB diagnostic facilities and intensifying active case findings using health extension workers is urgent issues to be addressed in order to effectively control the increasing trend of drug resistance TB in Ethiopia .M. tuberculosis and drug resistant strain. Thus we believe that the finding has highlighted the real burden of anti-TB drug resistance at community level. However the study is not without limitation. First, symptoms suggestive of TB were used as screening mechanism. The fact that chest X-ray was not used in the current study might have resulted in missed asymptomatic TB cases and underestimate the burden of the disease. Second, the current study excluded 20 contaminated sputum cultures which may have an impact on our result.To our knowledge, this is the first population-based study that analysed the prevalence of primary and secondary drug resistance and MDR-TB in Ethiopia. It might also be one of the very few studies conducted in poor resource setting. In fact, experienced and qualified laboratory technicians carried out the smear microscopy, sputum culture and DST to isolate The study has identified a high rate of primary and secondary resistance to any of the first-line anti-TB drugs and MDR-TB in the study area. The high rate of anti-TB drug resistance may have resulted from sub-optimal performance of DOTS programme in detecting infectious TB cases, and low cure rate in the study area. As anti-TB drug resistance is linked with pervious TB treatment, there is a need to strengthen DOTS and DOTS-Plus programme and expand MDR-TB diagnostic facilities so as to detect the cases in time and start appropriate treatment to prevent the spread of MDR-TB in Ethiopia.AFB, Acid-Fast Bacillus; AOR, adjuster odd ratio; CI, confidence intervals; DOTS, directly observed short course treatment; DST, drug susceptibility tests; EMB, ethambutol; HEW, health extension worker; HIV, Human Immunodeficiency Virus; INH, isoniazid; IUATLD, International Union against Tuberculosis and Gung Disease; LJ, Löwenstein-Jensen; MDR-TB, multi- drug-resistant TB; RIF, rifampicin; STM, streptomycin; TB, tuberculosis; WHO, World Health Organization; XDR-TB, extensively drug-resistant TB


These results suggested that the MMC-loaded polymer-lipid hybrid NPs might be useful and efficient drug delivery systems for widening the therapeutic window of MMC and bringing the clinical use of MMC one step closer to reality.Mitomycin C is one of the most effective chemotherapeutic agents for a wide spectrum of cancers, but its clinical use is still hindered by the mitomycin C (MMC) delivery systems. In this study, the MMC-loaded polymer-lipid hybrid nanoparticles (NPs) were prepared by a single-step assembly  of MMC-soybean phosphatidyhlcholine (SPC) complex  and biodegradable polylactic acid (PLA) polymers for intravenous MMC delivery. The advantage of the MMC-SPC complex on the polymer-lipid hybrid NPs was that MMC-SPC was used as a structural element to offer the integrity of the hybrid NPs, served as a drug preparation to increase the effectiveness and safety and control the release of MMC, and acted as an emulsifier to facilitate and stabilize the formation. Compared to the PLA NPs/MMC, the PLA NPs/MMC-SPC showed a significant accumulation of MMC in the nuclei as the action site of MMC. The PLA NPs/MMC-SPC also exhibited a significantly higher anticancer effect compared to the PLA NPs/MMC or free MMC injection  Nanotechnology represents a powerful tool in the field of medicine to deal with cancer,2, whichIn recent years, the polymer-lipid hybrid NPs designed to merge the best of both worlds had been reported in several groups. The first was that the polymer-lipid hybrid NPs were prepared via the fusion of the polymeric NPs and liposomes. The preMitomycin C (MMC) is a water-soluble anticancer and antibiotic agent by inhibiting DNA synthesis and nuclear division and has been extensively used to treat various cancer including stomach, liver, breast, pancreas, colon, and bladder cancers. MMC wasin vivo for several decades. SPC had always been regarded as a promising candidate for drug preparations due to the biocompatibility, biodegradability, metabolic activity, and low toxicity and cytotoxicity compared to synthetic alternatives[in vitro drug release, and in vitro and in vivo effectiveness was tested in H22 cells and H22 tumor-bearing mice.Previously Wang et al. prepared the polymer-lipid hybrid NPs for systemic delivery of small interfering RNA (siRNA). In thisrnatives,33,34. MN,N-dimethylformamide (DMF), 3-(2-benzothiazolyl)-N,N-diethylumbelliferylamine (coumarin-6), fluorescein 5-isothiocyanate (FITC), and Dulbecco's modified Eagle's medium (DMEM) were purchased from Sigma-Aldrich . Iodide [1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR) was provided by Molecular Probes Inc. . A dialysis bag  was ordered from Greenbird Inc. . Deionized (DI) water was used throughout. Fetal bovine serum (FBS) was purchased from Gibco Life Technologies . Trypsin-EDTA (0.25%) and penicillin-streptomycin solution were from Invitrogen . All solvents used in this study were high-performance liquid chromatography (HPLC) grade. Mouse hepatoma cell line H22 cells were provided by the Cell Bank at the China Academy of Science.All chemical reagents were of analytical grade and used without further purification unless otherwise stated. Mitomycin C  was purchased from Hisun Pharmaceutical Co., Ltd. . SPC was provided by Lipoid GmbH . Poly  was provided by Daigang BIO Engineer Co., Ltd. . Firstly, MMC and SPC were added to tetrahydrofuran by vigorous agitation in a 40°C water bath for 4 h. The orgIn addition, the micron-sized PLA NPs/MMC-SPC  could be formulated as follow: FITC conjugated MMC  was prepared for use as a fluorescence probe. And then, FITC-MMC-SPC complex was prepared in a same way as described above. Thirty-six milligrams of FITC-MMC-SPC complex and 200 mg of PLA were dissolved in 10 mL of DMF, accompanied by vigorous stirring. The mixture was dialyzed against 1,000 mL of DI water for 8 h. Then, the micron-sized PLA NPs/FITC-MMC-SPC were collected by centrifugation at 15,000 rpm at 4°C for 10 min, after that, the micron-sized PLA NPs/FITC-MMC-SPC were resuspended in DI water.18 column ; temperature, 25°C; and elution flow rate, 1.0 mL/min. The mobile phase for the determination of MMC was water-methanol . The detection wavelength for the determination of MMC was 362 nm. The drug encapsulation efficiency and drug loading content were calculated according to the formulaFor determination of drug encapsulation efficiency and drug loading content, the lyophilized PLA NPs/MMC-SPC were dissolved in DMF. The organic solution was filtered and submitted to HPLC analysis. The amount of MMC was assayed using a HPLC  system consisting of a Waters 2695 Separation Module  and a Waters 2996 Photodiode Array Detector  with the following conditions: chromatographic column, symmetry CThe average particle size, polydispersity index (PDI), and zeta potential of the suspension of the PLA NPs/MMC-SPC were performed by dynamic light scattering (DLS) using a Malvern Zetasizer Nano-ZS . Particle size was evaluated by intensity distribution. The morphology of the PLA NPs/MMC-SPC was visualized by SEM  operating at 20 kV and TEM  operating at 200 kV.The lyophilized PLA NPs/MMC-SPC were suspended in phosphate buffer solution (PBS) (pH 7.4) and incubated at 37°C for 48 h. The particle size was assayed at 12 h intervals by DLS.The release of MMC from the PLA NPs/MMC-SPC was determined by a dialysis technique. The lyophilized NPs were dispersed in 3 mL of PBS  buffer solution. The dispersions were transferred to a dialysis bag  and then subjected to dialysis against 10 mL of PBS at 37°C. At a predesigned time interval, all of the PBS buffer solution was withdrawn and subsequently replaced with the 10 mL of fresh PBS after each sampling. The PLA NPs/MMC and free MMC were used for comparison. The release of MMC was determined by a HPLC method as described above.22 cells (mouse hepatoma cell line) were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. The cells were cultivated in a humidified atmosphere containing 5% CO2 at 37°C.H22 cells were seeded at a density of 1 × 105 cells per well in 6-well plates with their specific cell culture medium. The cells were incubated at 37°C and 5% CO2 for 24 h. One-hundred microliters of the coumarin-6-PLA NPs/MMC-SPC and coumarin-6-PLA NPs/MMC at the equivalent coumarin-6 concentration was added and incubated further for 6 h. The cells were washed with PBS, fixed with 4% paraformaldehyde, and stained with Hoechst 33258 . The cells were observed using a Leica TCS SP5 confocal laser scanning microscopy .To facilitate the observation of cellular uptake, coumarin-6 was used as a hydrophobic fluorescence probe to load 22 cells were seeded in 6-well plates with a density of 2 × 105 cells/mL and incubated for 24 h, and then, the original medium was replaced with the coumarin-6-PLA NPs/MMC-SPC and coumarin-6-PLA NPs/MMC-SPC at the equivalent coumarin-6 concentration. The cells were incubated for the predesigned time at 37°C and then washed with cold PBS and harvested by 0.25% trypsin-EDTA. The harvested cells were suspended in PBS and centrifuged at 1,000 rpm for 5 min at 4°C. After washing and centrifugation, the cells were resuspended in PBS and performed by a Beckman Coulter EPICS XL flow cytometer .To further quantitatively investigate the cellular uptake, coumarin-6 was used as a fluorescence probe to load within the PLA NPs/MMC-SPC and PLA NPs/MMC, respectively . H22 cells, FITC conjugated MMC  was used to stain nuclei.To understand the intracellular distribution of the drug inside H linkage) was pre22 cells were exposed to the PLA NPs/MMC-SPC, PLA NPs/MMC or free MMC with different MMC concentrations for 24 h. The data were expressed as the percentage of surviving cells.The cytotoxicity of the drug delivery systems was measured using a MTT assay  according to the manufacturer's suggested procedures. HAll the animal procedures complied with the guidelines of the Xiamen University Institutional Animal Care and Use Committee. The experiments were performed on adult male SD rat weighing 200 ± 20 g (mean ± SD) from Shanghai Laboratory Animal Center. DiR was used as a near-infrared fluorescence probe to load within the PLA NPs/MMC-SPC and PLA NPs/MMC, respectively. One-hunin vivo imaging, 0.2 mL of the DiR-PLA NPs/MMC-SPC was injected into the nude mice bearing H22 tumor via the lateral tail vein. Imaging was performed at 1, 3, and 12 h after injection using a Maestro™ in vivo imaging system . At 12 h post-injection, the mice were sacrificed. The tumor and normal tissues  were excised, followed by washing the surface with 0.9% NaCl for the ex vivo imaging of DiR fluorescence using a Maestro™ in vivo imaging system . The mice treated without 0.2 mL of the DiR-PLA NPs/MMC-SPC were used for comparison.For 6 H22 cells in the right axillary region of the mice before the treatment. H22 tumors were not hormone dependent and easily grown in the subcutaneous layer of mice. The tumors were allowed to grow for 1 week after tumor transplantation, after that, the treatment was performed. The H22 tumor-bearing mice were randomly divided into 4 groups (10 mice per group): group 1 for 0.9% NaCl, group 2 for PLA NPs/SPC, group 3 for free MMC injection, and group 4 for PLA NPs/MMC-SPC. The mice were intravenously administrated at 4 mg/kg  every 2 days for 3 times. Each mouse was earmarked and followed individually throughout the whole experiments. The tumor size and body weight were then monitored every 2 days. The mice were euthanized on day 12. The greatest longitudinal diameter (length) and the greatest transverse diameter (width) of the tumor were measured using a vernier caliper, and the tumor volume was calculated using length × width2 × 0.5.Kunming mice aged 4 to 5 weeks  were supplied by Xiamen University Laboratory Animal Center and used in this study. Subcutaneous tumors were established in the mice by subcutaneous inoculation of 5 × 10In this study, a single-step dialysis method by addition of the amphiphilic MMC-SPC complex to the PLA polymer solution in DMF was used to form the hybrid PLA NPs/MMC-SPC. DMF is water miscible and a good solvent for the PLA polymer and MMC-SPC complex. PLA and MMC-SPC were first added to DMF (organic phase) and were then dialyzed against water (aqueous phase). After the exchange of the organic phase and the aqueous phase under continuous dialysis process, the PLA polymer and MMC-SPC complex precipitated to form the PLA NPs/MMC-SPC with a core-shell structure, and the amphiphilic MMC-SPC complex was adsorbed and self-assembled on the surface of the PLA core by hydrophobic interaction, as illustrated in Figure The hybrid PLA NPs/MMC-SPC comprised a hydrophobic PLA core and an amphiphilic MMC-SPC shell. Once intravenously administrated, the polymer-lipid hybrid NPs were accumulated at the tumor site via the enhanced permeability and retention (EPR) effect. And then, the polymer-lipid hybrid NPs were internalized by the cells via endocytosis. Lastly, MMC was released from the polymer-lipid hybrid NPs and delivered to the nuclei, presenting the anticancer activity.The effect of the MMC-SPC concentration on the size, zeta potential, and encapsulation efficiency of the hybrid PLA NPs/MMC-SPC was investigated. As shown in Additional fileTable As it is reported, a particle size (200 to 300 nm) may be suited for the EPR effect,40, provThe clinical use of the PLA NPs as intravenous drug delivery systems is limited due to their poor physiological stability. We thus investigated if the hybrid PLA NPs/MMC-SPC can overcome this drawback. As shown in Figure in vitro drug release profiles of the PLA NPs/MMC-SPC and PLA NPs/MMC were monitored as a function of time. As clearly shown in Figure The 22 cell as the model to evaluate the cellular uptake of the coumarin-6-labeled PLA NPs/MMC-SPC and PLA NPs/MMC at the equivalent concentration of coumarin-6 using confocal laser scanning microscopy. To better compare the cell internalization among the coumarin-6-PLA NPs/MMC-SPC and coumarin-6-PLA NPs/MMC, the images were taken by harmonizing the parameters such as laser power, sensitivity, offset, and gain constant during the cell imaging procedure. As shown in Figure 22 cells after 6 h of incubation, indicating the internalization of the PLA NPs/MMC-SPC and PLA NPs/MMC. More importantly, the significantly higher fluorescence intensity was observed in the cytoplasm of H22 cells treated with the coumarin-6-PLA NPs/MMC-SPC compared to those treated with the coumarin-6-PLA NPs/MMC with the same incubation time under the identical instrumental conditions. The result can be explained by the existence of SPC on the surface of the hybrid PLA NPs/MMC-SPC. Since SPC was an amphiphilic molecule composing of a hydrophilic head and a hydrophobic tail and also was similar to the phospholipids of the cell membrane in the structure component; thus, the cellular uptake of the hybrid PLA NPs/MMC-SPC was facilitated by the mutual interaction between the hybrid PLA NPs/MMC-SPC and the cell membrane.We selected H22 cells were incubated with the coumarin-6-PLA NPs/MMC-SPC and coumarin-6-PLA NPs/MMC and subsequently analyzed the cells by flow cytometry. As clearly shown in Figure To more precisely understand how the composition of formulations affected the cell internalization of the NPs, HConsistent with the observations by confocal laser scanning microscopy, the result of flow cytometry confirming that enhanced cellular internalization (45.6% increased for 4.5 h) of the PLA NPs/MMC-SPC compared with the PLA NPs/MMC. The lipophilicity and liposolubility of SPC on the surface of the hybrid PLA NPs/MMC-SPC perhaps increased the endocytosis and facilitated the passive delivery of the PLA NPs/MMC-SPC NPs to the interior of the cells.22 cells were treated with the PLA NPs/FITC-MMC-SPC, and the intracellular drug delivery is shown in Figure The fate of MMC after entering the cell is important for subsequent induction of cell death/apoptosis in the nuclei,45. To iFor both the PLA NPs/FITC-MMC-SPC and PLA NPs/FITC-MMC, the green fluorescence was presented in the cell. This finding was similar to the reported work. It was 22 cells incubated with the PLA NPs/FITC-MMC-SPC were significantly increased compared with those incubated with the PLA NPs/FITC-MMC at the same incubation time under the identical instrumental conditions, the result combined with the in vitro cellular uptake further confirmed that the interaction of SPC on the surface of the hybrid PLA NPs/FITC-MMC-SPC, and the cell membrane enhanced the cellular uptake of the nanoscaled drug delivery systems, hence, increased the intracellular internalization and accumulation of FITC-labeled MMC. A sufficient intracellular drug concentration was essential for an enhanced anticancer activity. Escape of the delivered MMC from endo/lysosome into nuclei was important as the target site of MMC was nuclear DNA and MMC could interrupt its function. Although more studies were needed to better understand the nuclear radiolabeled MMC delivery and the underlying mechanism, the result did suggest that our NPs were capable of effectively mediating intracellular drug delivery.It should be noted that, the fluorescence signals of H22 cells for 24 h. It was straightforward to understand that the lower cell viability corresponds to the higher concentration of drugs. Notably, the PLA NPs/MMC-SPC significantly reduced the cell viability compared to the PLA NPs/MMC at the same drug concentration. As it is reported, the drug-loaded polymer NPs might be internalized by endocytosis due to their nanoscaled particle size[The efficacy of the PLA NPs/MMC-SPC to defeat cancer cells is reflected by their cytotoxicity. As shown in Figure cle size. Neverth22 cells at an equivalent MMC concentration . Once accumulated at the tumor site, the polymer-lipid hybrid NPs as a whole would easily into the interior of the tumor cells to exert the pharmacological effects.Besides, the cytotoxicity PLA NPs/MMC-SPC were not as effective as the free MMC against H Figure , which w Figure . Such a in vivo stability of PLA NPs/MMC-SPC was evaluated by monitoring the blood clearance of the PLA NPs/MMC-SPC .Effect of the MMC-SPC concentration on the particle size, zeta potential, and drug encapsulation efficacy of the hybrid PLA NPs/MMC-SPC. Data are presented as mean ± SD (Click here for file


Drosophila melanogaster strains, which avoided shock to varying extents. We identified 514 genes whose expression levels and/ or sequences co-varied with shock avoidance scores. We independently scrutinized 14 of these genes using mutants, validating the effect of 7 of them on shock avoidance. This emphasizes the value of our candidate gene list as a guide for follow-up research. In addition, by integrating our association results with external protein-protein interaction data we obtained a shock avoidance-associated network of 38 genes. Both this network and the original candidate list contained a substantial number of genes that affect mechanosensory bristles, which are hair-like organs distributed across the fly’s body. These results may point to a potential role for mechanosensory bristles in shock sensation. Thus, we not only provide a first list of candidate genes for shock avoidance, but also point to an interesting new hypothesis on nociceptive mechanisms.Electric shock is a common stimulus for nociception-research and the most widely used reinforcement in aversive associative learning experiments. Yet, nothing is known about the mechanisms it recruits at the periphery. To help fill this gap, we undertook a genome-wide association analysis using 38 inbred  Drosophila melanogaster, which has well-known advantages when it comes to detecting gene-behaviour relationships, which are often conserved through evolution [Electric shock induces strong defensive and aversive behaviour in animals and is rated as painful by humans. Accordingly, it has become a traditional aversive reinforcement for associative learning research across species, including humans, e.g., –6. Althovolution –11.Instead of using artificial fly mutants, we took advantage of natural variation. We tested 38 nature-derived inbred fly strains in shock avoidance and then looked for associations between their behavioural scores and both gene expression level- and single nucleotide polymorphism (SNP)-data, which were already available ,13. ThisWith this strategy, we uncovered 514 candidate genes for shock avoidance. We independently scrutinized 14 of these genes using mutants and found shock avoidance-roles for 7 of them, corroborating the validity of our genome-wide approach. In addition, we integrated our association analysis results with existing protein-protein interaction data, revealing a shock avoidance-associated network of 38 genes. This network, as well as the original candidate list contained a substantial number of genes relevant for mechanosensory bristles—hair-like body-surface organs, potentially suggesting a role for these in the sensation of shock.P< 0.0001; for sex-specific scores and analyses, see 2 = 0.0002, P = 0.94; Males: R2 = 0.0080, P = 0.59, N = 38 in each case).We tested 38 wild-derived inbred fruit fly strains for their choice in a maze with one electrified and one non-electrified arm . The resP< 0.05/ 18 769, corresponding to a Bonferroni correction). We thus refrained from a family-wise error-rate calculation and considered the 588 cases with P< 0.05 to be suggestive associations . We considered the 607 cases with P< 0.0005 to be suggestive associations . Furthewww.flybase.org) [In order to explore the interactions between our candidate genes, we made use of a large, experimental evidence-based network featuring 5 280 genes and 63 796 pair-wise physical interactions between the encoded proteins (ase.org) . We assiase.org) , revealiwww.flybase.org) [nito, CG3862, regucalcin, CG8671) may now be attractive candidates for bristle-relevant function. Thus, although we cannot exclude the possibility that other, bristle-independent cellular functions of these genes may make them critical for shock avoidance, it is tempting to hypothesize a potential role for mechanosensory bristles in the sensation of shock. In support of this, among our 514 candidate shock avoidance genes, 91 were identified in mutagenesis and/ or RNAi screens as relevant for mechanosensory bristles  . Of thesAs in any other genome-wide association study, the present study must likewise take into account the possible inter-dependency of alleles at different polymorphic loci (i.e. linkage disequilibrium) and, similarly, the inter-correlations between expression levels of different genes . In otheCG3711, rad50 and CG15107, the respective mRNA levels were also reduced in the mutant as revealed by real time quantitative RT-PCR (CG13397 the mutants tended to avoid shock less strongly than the controls (CG13397 gene (P< 0.05/ 3 and N = 28 in each case).For independent testing, we chose 14 candidate genes which were associated with shock avoidance in terms of expression level . These had been generated by full-sib inbreeding of iso-female strains from Raleigh, North Carolina, USA for more than 20 generations [white1118-mutant strains used for generating the respective transposon insertion mutant-collections . This resulted in three independent cases of precise excision of the transposon, resulting in a wild-type locus (CG13397 (2L: 8411078 to 2L: 8411653)  .All experiments took place at 21–23°C temperature, 60–70% relative humidity under bright fluorescent light. Flies were tested in groups of ~ 50. At 0:00 min , flies wWe designed a locomotion assay that was directly comparable to the above-described shock avoidance assay in that it employed the same setup and had the same temporal flow. Following www.r-project.org) on a PC. We used non-parametric statistics: Kruskal-Wallis tests for global and Mann-Whitney U-tests for pair-wise comparisons. In Figs P-value by the number of tests (i.e. Bonferroni correction).Behavioural scores were analyzed using Statistica version 11.0  and R version 2.15.1 (www.r-project.org) was used for these analyses. Raw Affymetrix GeneChip Drosophila Genome 2.0 expression microarray data [www.ebi.ac.uk/arrayexpress (accession number EMEXPE-MEXP-1594) using the R Affy package [R version 2.15.1 (ray data  for the  package . The raw package . For eacP< 0.05 were considered to be associated with shock avoidance (see www.affymetrix.com) and the FlyBase [www.flybase.org). To determine the candidate genes, we excluded the probe-sets with ‘_x_’ or ‘_s_’ qualifiers in their probe-set Affymetrix IDs, as these contain one or more probes that hybridize with products of different genes. Those genes for which at least one corresponding probe-set fulfilled the statistical criterion for association were considered to be candidates (β0 was the intercept and β1 the estimate for the effect of the mean expression level. β1 was compared to zero with a two-tailed t-test (d.f. = 356). The probe-sets with a  FlyBase   with minor allele frequency (MAF)> 0.1  and call-rate> 0.7. For each such SNP, we tested for an effect on the shock avoidance scores using the following linear model:The Illumina and 454 SNP calls of the 38 inbred strains  were dowP< 0.0005 were considered to be suggestive associations (see P< 0.0005 in at least one sex were taken as associated with shock avoidance (see Drosophila melanogaster reference genome version 5.35 and the FlyBase [www.flybase.org). For defining the candidate genes, those SNPs that were annotated to multiple genes were excluded and those genes that had at least one SNP fulfilling the statistical criterion for association were taken as candidates  [To probe for the enrichment of GO terms for Biological Process, Cellular Compartment and Molecular Function, we analyzed our list of 514 candidate shock avoidance genes  against ome.jsp) ,36 with www.bioconductor.org) and the Batch Processing Tool of FlyBase [www.flybase.org). On the other hand, the protein—protein interaction data were downloaded from FlyBase . From the intersection of these two datasets a network of 5 280 genes and 63 796 pair-wise interactions was derived. For each gene in this network, the P value for the association between the expression level and the shock avoidance scores was converted into a network node score based on the negative decadic logarithm of the P value. Subsequently, these scores were adjusted using a P value cut-off of 0.025, such that only P values smaller than this threshold were positively scored, while the remaining non-significant P values obtained negative scores. Using an integer linear programming formulation, the optimally scoring sub-module was calculated exactly [This analysis followed up on the gene expression level—shock avoidance associations. On the one hand, for each of the 18 769 probe-sets considered for association, we obtained gene FlyBase IDs, using the R Bioconductor package drosophila2.db . The small differences in sample sizes across sexes arose because the shock avoidance scores calculated on the basis of less than 5 individual flies were excluded from analysis. Box plots as in The data from (TIF)Click here for additional data file.S2 FigA, each panel shows for the indicated gene the gender-specific Delta CT values of mutant vs. control in a scatter plot. For example, as in the case of CG15107 females, if the control had a median Delta CT of ~ 7, while the mutant had ~ 10, this indicated that the mutant mRNA level was ~ 2(7–10) = 0.125th of the control. In B, these fold change values  [Tsp42Ei, CG16865 and CG3290, reductions in the respective mRNA levels were found in the mutants, although shock avoidance was comparable to the controls Click here for additional data file.S3 FigTrained odour—#Control odour) 100 / #Total, where # indicates the number of flies in the respective maze-arm. Two subgroups of flies were always trained in parallel, switching the roles of two chemicals as control and trained odour. We used the odours 3-octanol  and benzaldehyde . A learning index was calculated based on the preferences of these two groups, in order to cancel out non-associative effects. Learning index = (PREFBA-Shock + PREFOCT-Shock) / 2, where the subscripts of PREF indicate the respective odour-shock contingency. Thus, negative learning indices indicated conditioned avoidance from the trained odour, whereas positive values indicated conditioned approach. B. A transposon insertion mutant of CG3711 performed worse than its corresponding control not only in shock avoidance . Box plots as in A. For the shock-reinforced olfactory learning assay, flies entered the setup at 0:00 min and were presented with a control odour from 4:00 min on for 15 s. A trained odour was in turn applied from 7:15 min on for 15 s, immediately followed by electric shock . At 12:00 min, flies were transferred to food vials to rest until they were re-introduced into the setup at 28:00 min. At 33:00 min, they were brought to the mid-point of a maze with two arms scented with either odour and were allowed to choose for 2 min. At the end of the choice, the maze-arms were sealed and the flies were counted to calculate an odour preference score as PREF = Click here for additional data file.S1 TableP values refer to the results of a two-tailed t-test comparing β1 to zero. We list probe-sets with P < 0.05. Annotations are based on Affymetrix documentation (www.affymetrix.com) and the FlyBase (www.flybase.org) [For each probe-set, we tested for a linear regression between the mean expression levels and the median unisex shock avoidance scores. β1 is the respective estimate for the effect of the expression level on shock avoidance. Negative β1 values indicate that the higher the expression level was, the stronger the shock avoidance was; positive β1 values reflect the converse, i.e., the higher the expression level, the weaker the shock avoidance. The t and (XLSX)Click here for additional data file.S2 TableP values refer to the results of a two-tailed t-test comparing β1 to zero. We list SNPs with P< 0.0005. Annotations are based on Drosophila melanogaster reference genome version 5.35 and the FlyBase [For each bi-allelic autosomal SNP with a favourable minor allele frequency and call rate, we tested for a linear regression between the allele type and the median unisex shock avoidance scores. β1 is the respective estimate for the effect of allele type on shock avoidance. The t and (XLSX)Click here for additional data file.S3 TableP< 0.0005 in at least one gender. Annotations are based on Drosophila melanogaster reference genome version 5.35 and the FlyBase [For each bi-allelic X-chromosome-linked SNP with a favourable minor allele frequency and call rate, we tested for a linear regression between the allele type and either the female or the male median shock avoidance scores. β1, t, and P are as explained for (XLSX)Click here for additional data file.S4 TableWe list the candidate shock avoidance genes revealed by expression level- and/ or SNP-associations from  Tables. (XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 TableBy superimposing the results of our gene expression level—shock avoidance analyses on the existing protein-protein interaction network, we obtained a shock avoidance-relevant network of 38 genes, which are listed along with the statistics of their association with shock avoidance (see the legend of (XLSX)Click here for additional data file.S7 TableFor 14 candidate genes from (XLSX)Click here for additional data file.S1 TextDetailed methodology for PCR and real-time quantitative PCR are given, along with a reference list for these as well as the Supplemental Tables and Supplemental Figures.(DOCX)Click here for additional data file.


For the detection of malignant mesothelioma no single biomarker with reasonable sensitivity and specificity has been described so far. Mesothelin, the most prominent blood-based biomarker, is characterized by high specificity but low sensitivity. It might be reasonable to combine biomarkers of different molecular classes in order to improve the overall performance. The aim of this study was to assess the performance of the combination of mesothelin and miR-103a-3p as blood-based biomarker for mesothelioma.Mesothelin concentration in plasma and miR-103a-3p levels in the cellular blood fraction were analyzed in 43 male mesothelioma patients and 52 male controls formerly exposed to asbestos. For the discrimination of epithelioid and biphasic mesothelioma from asbestos-exposed controls mesothelin and miR-103a-3p showed 74% and 89% sensitivity and 85% and 63% specificity, respectively. For the combination of mesothelin and miR-103a-3p a sensitivity of 95% and a specificity of 81% were calculated.The results of this study show that the combination of mesothelin and miR-103a-3p improves the diagnostic performance of individual blood-based biomarker to detect malignant mesothelioma. The obtained results indicate that the use of biomarkers of different molecular classes might be a reasonable approach to assemble a biomarker panel. Malignant mesothelioma is an aggressive cancer of the serous membranes with increasing incidence worldwide For the detection of cancer at early stages blood-based biomarkers are, in principal, a feasible approach, either for direct diagnosis of the disease or in order to guide suspicious cases to more costly diagnostic methods like High Resolution Computed Tomography (HRCT). Screening for mesothelioma might be clinically important in at-risk collectives composed of subjects formerly exposed to asbestos In recent years, two molecular classes were in focus as blood-based biomarkers for malignant mesothelioma, proteins and microRNAs (miRNAs). Several candidate biomarkers of both molecular classes were described, e.g., mesothelin Up to date, the most prominent biomarker for mesothelioma is mesothelin. However, mesothelin as an individual biomarker is characterized by a high specificity of 89%  but a relatively low sensitivity of 58%  for the discrimination of mesothelioma patients and asbestos exposed subjects as recently shown in a meta-analysis The aim of this study was to assess the combination of the protein marker mesothelin in plasma and the miRNA miR-103a-3p in the cellular blood fraction in order to enhance the biomarker performance to diagnose malignant mesothelioma.All participants of the study provided written informed consent. The study was designed according to rules guarding patient privacy and with the approval from the ethics committee of the Ruhr-Universität Bochum (reference number 3217–08).The study group consisted of 43 male patients with diagnosed mesothelioma including 28 epithelioid (65%), six biphasic (14%) and five sarcomatoid (12%) mesothelioma. The histological subtype was not specified in four cases (9%). None of the patients were treated by surgery, chemotherapy, or radiation therapy before blood collection. The control group consisted of 52 male subjects formerly exposed to asbestos. Detailed characteristics of the study groups are listed in From each participant peripheral blood was collected in 9.0 ml S-Monovette EDTA gel tubes  and centrifuged at 2,000×g for ten minutes at room temperature within 30 minutes after blood collection. Plasma was separated from the cellular fraction and both matrices were frozen immediately.Plasma mesothelin was measured using the ELISA kit MESOMARK  as described previously RNA isolation from 0.5 ml of the cellular fraction was performed using the RiboPure-Blood Kit according to the Alternate Protocol: Isolation of Small RNAs . Concentration of RNA was quantified by measuring the absorbance at 260 nm using a NanoDrop ND-100 spectrophotometer .vs. controls formerly exposed to asbestos and vs. healthy volunteers of the general population, whereas miR-125a was the most stable miRNA in the analyzed study groups -dCt expression values were used for statistical analysis. Raw Ct values are presented in TaqMan miRNA Assays (Life Technologies) were used for quantitative miRNA expression analysis of miR-103a-3p as biomarker and miR-125a as reference. In an earlier study of our group using oligonucleotide microarrays to analyze 328 miRNAs, miR-103a-3p was identified as significantly deregulated in mesothelioma patients PROC LOGISTIC in SAS was used to determine sensitivities and specificities of mesothelin and miRNA-103a-3p from receiver operating characteristic (ROC) curves illustrating the performance of both biomarkers and their combination to discriminate the studied groups. The ROC curves of the biomarker combination were calculated with mesothelin and miRNA-103a-3 as independent variables in a multiple logistic regression model. The bootstrap procedure  was used for internal validation of the estimates in the ROC analyses. Biomarker cut-offs were determined with maximum Youden's index (YI) or a false-positive rate (FPR) of 4%. Logistic regression was performed to calculate odds ratios (OR) of a false-positive test in controls and of a false-negative test in mesothelioma cases based on potential influencing factors.Statistical analyses were performed using SAS/STAT and SAS/IML software, version 9.3 . Box plots with median and inter-quartile range (IQR) were used to depict the distribution of mesothelin and miR-103a-3p. Groups were compared using the non-parametric Wilcoxon rank-sum test. Potential factors influencing the biomarkers were evaluated using multiple linear regression models with log-transformed biomarker values. Effect estimates were given as exp(β) with 95% confidence intervals (CI) and p values. Values of exp(β)>1 and <1 indicate a positive and a negative association between analyzed factor and mesothelin or miRNA-103a-3p, respectively. Distribution of mesothelin concentrations and normalized miR-103a-3p levels in the two study groups are shown in vs. epithelioid (p = 0.017) and sarcomatoid vs. biphasic mesothelioma (p = 0.036). Normalized miR-103a-3p levels showed a median value of 265.2 (IQR 141.6–614.9) for epithelioid, 179.0 (IQR 145.2–440.2) for biphasic, and 545.8 (IQR 342.2–1133.5) for sarcomatoid mesothelioma (Mesothelin showed a median concentration of 2.01 nmol/l (IQR 1.24–4.99 nmol/l) for epithelioid, 2.01 nmol/l (IQR 1.58–4.18 nmol/l) for biphasic, and 0.75 nmol/l (IQR 0.69–1.32 nmol/l) for sarcomatoid mesothelioma . Differethelioma . DiffereThe impact of study group characteristics as potential influencing factors on mesothelin and miRNA-103a-3p are shown in Excluding sarcomatoid mesothelioma from analysis revealed a median mesothelin concentration of 2.03 nmol/l (IQR 1.24–5.82 nmol/l) and a median miR-103a-3p value of 228.5 (IQR 144.2–608.5) for mesothelioma patients. Differences between mesothelioma patients and asbestos-exposed controls were still statistically significant for both biomarkers (p<0.001).Using ROC analyses including all subjects AUCs of 0.81 for mesothelin  A, 0.76 Sensitivities and specificities for mesothelin and miR-103a-3p are shown in Combination of mesothelin and miR-103a-3p resulted in continuously higher sensitivities and consistently high specificities . Using mROC analyses of 1,000 bootstrap samples resulted in similar sensitivities and specificities for the combination of mesothelin and miR-103a-3p in comparison to the original analyses . The calMesothelioma is an aggressive cancer, commonly diagnosed at late stages of the disease. Reliable blood-based biomarkers could improve detection of mesothelioma at early stages In this study mesothelin was combined with miR-103a-3p, resulting in an improved AUC of 0.93. According to the maximum YI a sensitivity of 95% and a specificity of 81% was calculated. Based on a FPR of 4%, representing 96% specificity, the sensitivity was 74%. The obtained results are based on relatively small study groups but bootstrap analysis with 1,000 random samples showed that the calculated sensitivities and specificities remained stable. Thus, it might be reasonable to test the combination in larger study groups.In recent years, several analyses were performed combining mesothelin with a number of different proteins. But none of the tested combinations showed a sufficient improvement of the biomarker performance. Whereas no advancement was shown for the combination of mesothelin with CA125 The combination of mesothelin and miR-103a-3p appears to be more complementary than a combination of biomarkers within the same molecular class. This might be due to differences between the mechanisms how biomarkers are released into blood. Generally, proteins are released during natural secretion processes, which can be altered by cancer. In contrast, DNA and RNA are mostly released during apoptosis, necrosis, and other processes. Therefore, a complementary effect between biomarkers of different molecular classes should be more likely and might result in an enhanced discrimination between cases and controls. Also, miR-103a-3p was not isolated from plasma but from the cellular fraction of blood. Thus, it has to be considered that changes in levels of miR-103a-3p may be the result of an indirect effect, i.e., a response of the immune system to the tumor.GPRC5A mRNA and its protein product in pancreatic cells GPRC5A acts as oncogene or tumor suppressor in different types of cancer GPRC5A in mesothelioma. Thus, it might be reasonable to evaluate the potential roles of miR-103a-3p and GPRC5A in mesothelioma.For mesothelin a role in cell adherence, proliferation, and cancer progression has been implied downregulated in the blood of mesothelioma patients Using immunohistochemistry, breast carcinoma, particularly triple-negative breast cancer that metastasize to the pleura and lung, might be confused with mesothelioma The use of individual biomarkers for diagnosis is frequently characterized by relatively low sensitivities and/or specificities. This is also true for miR-103a-3p and mesothelin alone. Generally, high specificities are required in cancer screening It is well known that mesothelin fails to detect sarcomatoid mesothelioma The knowledge of biological, pre-analytical, and analytical factors influencing the biomarker levels is important for the assessment of the reliability of a biomarker For translational medicine it is essential that potential biomarkers are measurable in routine clinical practice. For mesothelin it was shown that the used ELISA is applicable for the measurement on automated systems In conclusion, we showed that the combination of mesothelin and miR-103a-3p improved the diagnostic performance of a blood-based screening test, resulting in higher sensitivity and specificity to detect malignant mesothelioma. The results support the concept that combinations of rather than individual biomarkers are needed for a reliable diagnosis of malignant diseases. In addition, it seems especially promising to use combinations of markers from different molecular classes, i.e. proteins and nucleic acids, to improve diagnostic performance.Table S1Subjects characteristics, raw data of miR-103a-3p and miR-125a expression analysis, and mesothelin concentration.(XLSX)Click here for additional data file.


Objective: In this study, the effects of estradiol valerate and raloxifenea selective estrogen receptor modulator; (SERM) on morphine induced sensitization were examined in mice memory, according to the step-down passive avoidance task.Method: The mice received morphine or estradiol and raloxifene for three days alone or in combination with morphine. After a drug free period of 5 days, the subjects received saline or morphine as pre- training treatments followed by a pre-test saline administration. The memory retrieval was evaluated using step-down passive avoidance test both on the training and test day.Results: The results illustrated that the three- day administration of morphine induced sensitization through the enhancement of memory retrieval (morphine induced sensitization in mice memory). Both the three- day administration of estradiol valerate alone and with morphine (5 mg/kg) restored memory. On the other hand, the three- day administration of raloxifene had no effect on memory retrieval alone, but declined morphine induced sensitization in mice memory.Conclusion: The results of the study indicated that there is an interaction between estrogen receptor modulators and morphine induced sensitization in mice memory. Large body of evidence indicate that abuse of such drugs as morphine affects neuronal plasticity in brain areas related to motivation and reward . BesidesIt has been proved that several effects of acute and chronic exposure to morphine are expressed differently on the basis of gender such as anti-nociception , locomotAccording to previous reports, estrogen plays a considerable role in induction of acute tolerance to morphine induced analgesia . MoreoveThe purpose of this study was to evaluate the effects of various doses of estradiol valerate and raloxifene  on morphine induced sensitization in mice memory.AnimalsMale adult NMRI mice  weighing 20.2–29.4 g were used in the present study. The animals were housed in a temperature/moisture controlled (22±3°C/45-55% humidity) colony room and were maintained in a 12-h light/dark cycle with free access to food and water, except during experiments. Experiments were done between 10:00 a.m. and 3:00 p.m. Animals were adapted to the laboratory conditions for at least 72 hours prior to experiments. Each treatment group included ten animals. The protocols were carried out according to national guidelines for animal care and use which was approved by the Ethics Committee of the institute.Drugs and Chemicals:Morphine sulphate was purchased from Temad (Iran). Estradiol valerate, raloxifene and ultra- filterated sesame oil (as a vehicle for estradiol) were purchased from Iran Hormone Company. Polyethylene glycol 300 (PEG300) was purchased from Merck Schuchardt OHG .Morphine sulfate was dissolved in normal saline (0.9%) and estradiol valerate emulsified in sesame oil and normal saline (0.9%). PEG300 was used as a vehicle for raloxifene.Passive Avoidance ApparatusThe passive avoidance apparatus includes a wooden box (30×30×40 cm height), the floor of which is made of 29 parallel stainless steel bars . A wooden platform (4×4×4 cm) is set in the center of the grid floor. Intermittent electric shocks  were transferred to the grid floor by an insulated stimulator . A single-trial step-down passive avoidance task was accomplished applying this apparatus. Each mouse was gently placed on the wooden platform. When the mouse stepped-down from the platform and placed all its four paws on the grid floor, then it received electric shock for 15 sec. For establishing the retention test, each mouse was placed on the platform again at 24 h following training and the step-down latency was recorded with a stopwatch. An upper cut-off time of 300 sec was allocated for time recording.ExperimentsExperiment 1: This experiment examined morphine induced sensitization in passive avoidance memory. Animals in one control group received 10 ml/kg normal saline subcutaneously (S.C) both in pre-training and pre-test administrations. The other control group received 5 mg/kg (S.C) morphine as pre-training and saline as a pre-test treatment. Three other groups received 5, 10 and 20 mg/kg morphine intraperitoneally (I.P) for three days; after five days of drug-free period, they received 5 mg/kg morphine as pre-training followed by a pre-test administration of saline .Experiment 2: This experiment assessed the role of estradiol valerate on morphine induced sensitization in learning. In these experiments all animal groups received morphine  as pre-training and saline (10 ml/kg) as pre-test treatment. One set of animal groups received 0.45, 0.9 and 1.8 mg/kg estradiol valerate intraperitoneally (I.P) with a concomitant saline administration  for three days. The other set of groups received 0.45, 0.9 and 1.8 mg/kg estradiol valerate (I.P) with a concomitant morphine administration  for three days.Experiment 3: This experiment evaluated the role of raloxifene on morphine induced sensitization in learning. In this experiment, animal groups received 10 ml/kg saline or 5 mg/kg morphine (S.C) as pre-training and saline (10 mg/kg) as a pre-test treatment. One set of animal groups received 5, 10 and 20 mg/kg raloxifen (I.P), followed by saline administration  for three days. The other set received 5, 10 and 20 mg/kg raloxifen (I.P) and morphine  for three days.Data AnalysisThe step-down latencies were expressed as the median and interquartile range. Data were analyzed using Kruskal–Wallis non-parametric one-way analysis of variance (ANOVA), followed by two-tailed Mann–Whitney U-test completed by a Holm’s Bonferroni correction for the paired comparisons to evaluate the significance of the results. In all statistical evaluations P<0.05 was used as the criterion for statistical significance.The results illustrated that pre-training administration of morphine (5 mg/kg) impaired the memory retrieval on the test day compared to the saline-treated group , which was restored in groups which received different doses of morphine for three days  (morphine induced sensitization in memory) = 29.24, p < 0.001, Mann Whitney U-test, P = 0.003 & 0.00003 for 10 and 20 mg/kg of morphine) .As shown in The results of this study revealed that pre-training administration of morphine (5 mg/kg) impaired the memory retrieval on the test day. Results from previous studies on opioids role in memory are notorious; it has been shown that spatial memory and synaptic plasticity  29) has has29) hThe results obtained from morphine induced sensitization in mice learning illustrated that the three-day administration of morphine  restored memory impairment by pre-training administration of morphine (5 mg/kg). These findings confirm the sensitization in learning induced by morphine which was first introduced in 2000  and lateMoreover, the results indicated that administration of estradiol valerate for three days instead of morphine, enhanced memory retrieval which was impaired previously by pre-training morphine. Several evidences propose that the most important gonadal steroid hormone (17ß-estradiol) in females may have a positive effect on memory and learning such as motor skills and spatial memory. Potentiating cerebellar plasticity and synapse formation in motor skills have been suggested as mechanisms involved in memory and learning. It has been reported that at least one of the estrogen receptors  in the hippocampus involves in spatial memory enhancement , 40. LatOn the other hand, the present study showed that co-administration of estradiol valerate with morphine, improved morphine induced sensitization in mice learning . Plenty In the last experimental set of this study, the three- day I.P injection of raloxifene alone represented no significant effect on memory retrieval. According to previous studies, the effects of raloxifene on memory are paradoxical. In other words, it has been demonstrated that raloxifene did not damage cognition or affect mood in postmenopausal women , but braIn this study, a three- day protocol of co-administrating raloxifene with morphine diminished morphine induced sensitization in mice learning in contrast to estradiol. There are little data regarding raloxifene (or SERM) and morphine interaction particularly memory related phenomena. In addition, it is revealed that tamoxifen was able to disturb consolidation and retrieval of morphine-associated contextual memory and this impairing effect might be inhibited by estradiol treatment . Also, iThe present study was done with a SERM  which a full estrogen receptor antagonist like fulvestrant was better than instead.The results of the present study revealed an interaction between estrogen receptor modulators and morphine induced sensitization in mice learning.